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United States Patent |
6,235,775
|
Sato
,   et al.
|
May 22, 2001
|
Acetone adduct of fungicidal V-28-3M
Abstract
The present invention provides an acetone adduct of V-28-3M to be used as
an intermediate for producing V-28-3M which is useful as a fungicide, a
process for producing it and a process for purifying V-28-3M. When crude
V-28-3M is dissolved in acetone, an acetone adduct of V-28-3M is to
remarkably improve the crystallization rate and, accordingly, also to
remarkably improve the purity. The obtained acetone adduct can be easily
hydrolyzed to obtain pure V-28-3M.
Inventors:
|
Sato; Yutaka (Kawasaki, JP);
Aoki; Yuuichi (Kawasaki, JP)
|
Assignee:
|
Ajinomoto Co., Inc. (Tokyo, JP)
|
Appl. No.:
|
525320 |
Filed:
|
March 13, 2000 |
Foreign Application Priority Data
Current U.S. Class: |
514/460; 549/415 |
Intern'l Class: |
C07D 315/00; A61K 031/35 |
Field of Search: |
514/460
549/415
|
References Cited
U.S. Patent Documents
4177265 | Dec., 1979 | Michel et al.
| |
5023079 | Jun., 1991 | Komoda et al.
| |
Foreign Patent Documents |
4-12199 | Apr., 1992 | JP.
| |
4-53878 | Aug., 1992 | JP.
| |
5-59084 | Mar., 1993 | JP.
| |
Primary Examiner: Lambkin; Deborah C.
Attorney, Agent or Firm: Oblon, Spivak, McClelland, Maier & Neustadt, P.C.
Parent Case Text
This application is a Continuation of International application No.
PCT/JP98/04070 Filed on Sep. 10, 1998.
Claims
What is claimed is:
1. Acetone adduct of V-28-3M represented by formula (1)
##STR3##
2. A process for producing the acetone adduct according to claim 1,
comprising the steps of dissolving a material containing V-28-3M
represented by following formula (2) in acetone, concentrating and/or
cooling the obtained solution to precipitate and to separate impurities
from the solution, and concentrating and/or cooling the mother liquor to
precipitate the crystals of the acetone adduct according to claim 1
##STR4##
3. The process according to claim 2, wherein the V-28-3M-containing
material is dissolved in acetone at a temperature in the range of
20.degree. C. to the boiling point of acetone to obtain a solution having
a concentration of 0.1 to 10 g/l, the solution is cooled to 10.degree. C.
or below to precipitate and thereby to separate impurities from the
solution, the mother liquor is concentrated to a concentration of 1 to 20
g/l, seed crystals are added to the obtained concentrate, and the
resultant mixture is cooled to 20.degree. C. or below to form crystals of
the acetone adduct.
4. The process according to claim 2, wherein the V-28-3M-containing
material is dissolved in dimethyl sulfoxide or dimethylformamide, a first
organic solvent is added to the obtained solution to precipitate and
thereby to separate impurities from the solution, and a second organic
solvent having a lower polarity than the first organic solvent is added to
the mother liquor to precipitate V-28-3M to be used as the
V-28-3M-containing material.
5. The process according to claim 4, wherein the V-28-3M-containing
material is dissolved in dimethyl sulfoxide or dimethylformamide at room
temperature to obtain a solution having a concentration of 1 to 100 g/l.
6. The process according to claim 4, wherein the first organic solvent is
selected from the group consisting of ethanol, isopropyl alcohol,
n-butanol, ethyl acetate and isopropyl acetate.
7. The process stated according to claim 4, wherein the second organic
solvent is selected from the group consisting of n-hexane, n-heptane and
toluene.
8. A process for purifying V-28-3M, comprising the steps of dissolving a
material containing V-28-3M represented by formula (2) in acetone,
concentrating and/or cooling the obtained solution to precipitate and
thereby to separate impurities from the solution, concentrating and/or
cooling the mother liquor to form crystals of the acetone adduct according
to claim 1, and dissolving the obtained crystals in a water containing
organic solvent to hydrolyze the acetone adduct crystals and thereby to
obtain V-28-3M
##STR5##
9. The purification process according to claim 8, wherein a solvent
selected from the group consisting of water containing methanol, ethanol,
isopropyl alcohol and acetonitrile is used as the water containing
solvent.
10. The purification process according to claim 9, wherein the water
containing solvent contains a weak acidic salt having a buffering effect.
11. The purification process according to claim 10, wherein the weak acidic
salt having the buffering effect is selected from the group consisting of
ammonium acetate, sodium acetate and sodium phosphate.
12. The purification process according to claim 8, comprising the step of
dissolving the V-28-3M-containing material in dimethyl sulfoxide or
dimethylformamide, adding a first organic solvent to the obtained solution
to precipitate and thereby to separate impurities from the solution, and
adding a second organic solvent having a lower polarity than the first
solvent to the obtained mother liquor to precipitate V-28-3M.
13. The purification process according to claim 12, wherein the
V-28-3M-containing material is dissolved in dimethyl sulfoxide or
dimethylformamide at room temperature to obtain a solution having a
concentration of 1 to 100 g/l.
14. The purification process according to claim 12, wherein the first
organic solvent is selected from the group consisting of ethanol,
isopropyl alcohol, n-butanol, ethyl acetate and isopropyl acetate.
15. The purification process according to claim 12, wherein the second
organic solvent is selected from the group consisting of n-hexane,
n-heptane and toluene.
Description
BACKGROUND OF THE INVENTION
The present invention relates to an intermediate for V-28-3M useful as a
fungicide, a process for producing it and a process for purifying V-28-3M
through the intermediate.
V-28-3M has a high fungicidal activity and a low toxicity. In a previously
known method for the purification of V-28-3M in the form of a crude
precipitate thereof, after an acid amide or urea is added to an aqueous
solution thereof or a solution thereof in a water containing solvent, an
insoluble material is removed and an alkali is added to the supernatant to
precipitate V-28-3M [Japanese Patent Unexamined Published Application
No.5-59084](hereinafter referred to as JP-Kokai 5-59084). However,
although the purity of V-28-3M was increased, the crystallization thereof
was difficult in this method, and for the further purification, only
reversed phase HPLC or counter current distribution method could be
employed [Japanese Patent Publication for Opposition Purpose No.4-53878
(hereinafter referred to as JP-Kokai 4-53878)].
In the reversed phase HPLC or counter current distribution method for the
high purification, the cost of the purification of the intended product is
high because an extremely large amount of the solvent is required for the
purification and the quantity of the crude product to be treated each time
is only small. Further, the increase in the scale of the laboratory is
difficult. Thus, this method is industrially not preferred.
V-28-3M is easily soluble in dimethyl sulfoxide or dimethylformamide, but
it is generally difficult to crystallize it from such a polar solvent in
which it is highly soluble. Also, although V-28-3M is easily soluble in a
water containing solvent such as an aqueous methanol solution, the
crystallization thereof from such a solvent is difficult. Methods of
recovering V-28-3M from a solution thereof in such a polar solvent in the
prior art include a method wherein an extremely large amount of water, for
example, 20 parts by volume per part by volume of the solution, is added
to the solution, taking advantage of the fact that V-28-3M is practically
insoluble in water, or a method wherein an alkali is added to the solution
to precipitate V-28-3M at a pH of 12 or above (JP-Kokai 5-59084). However,
the precipitate thus obtained could not be crystallized and, therefore,
the purity thereof did not reach to the level of that obatined by the HPLC
purification method.
SUMMARY OF THE INVENTION
The object of the present invention is to provide an acetone adduct of
V-28-3M from which highly pure V-28-3M can be obtained in a high yield.
Another object of the present invention is to provide a process for
producing the acetone adduct of V-28-3M.
Still another object of the present invention is to provide a process for
purifying V-28-3M by the crystallization to obtain highly pure V-28-3M in
a large scale, thereby to make up for the low productivity thereof.
After intensive investigations on the crystallization of V-28-3M, the
inventors have found that although V-28-3M is scarcely soluble in ordinary
organic solvents, it is slightly soluble in some solvents such as acetone
and methanol, particularly in acetone, the crystallization rate of V-28-3M
can be remarkably improved and, therefore, the purity thereof can be
remarkably increased because the amino sugar moiety of V-28-3M is
dehydration-condensed with acetone to form the acetone adduct. The
inventors have also found that since the obtained acetone adduct is easily
hydrolyzed in an aqueous solution to release acetone, purified V-28-3M can
be easily obtained.
In some cases, crude precipitates containing V-28-3M are contaminated with
high-molecular substances formed in the course of the culture, which
substances inhibit the dissolution of V-28-3M in acetone to lower the
recovery rate and also to inhibit the crystallization of the acetone
adduct. It has been found that the crystallization of the acetone adduct
can be facilitated if V-28-3M precipitate which may be obtained by
dissolving the crude precipitate in dimethyl sulfoxide or
dimethylformamide and adding an organic solvent to the resulting solution
to precipitate only the high-molecular substances, and removing the
precipitate, prior to the precipitation of V-28-3M by adding a solvent
having lower polarity is used as a starting material for the aceton
adduct, because high-molecular substances are removed from the crude
precipitate.
The present invention has been established on the basis of these findings.
Namely, the present invention provides a new acetone adduct of V-28-3M
represented by following formula (1):
##STR1##
The present invention also provides a process for producing the new acetone
adduct of V-28-3M represented by above formula (1), comprising the steps
of dissolving a material containing V-28-3M represented by following
formula (2) in acetone, concentrating and/or cooling the obtained solution
to precipitate and separate impurities from the solution, and
concentrating and/or cooling the mother liquor to precipitate the crystals
of new acetone adduct of V-28-3M:
##STR2##
The present invention also provides a process for purifying V-28-3M,
comprising the steps of dissolving the crystals of the acetone adduct of
V-28-3M obtained by the precipitation in the above-described production
process, dissolving the obtained crystals in a water containing organic
solvent to hydrolyze the acetone adduct crystals and thereby obtaining
V-28-3M.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a powder X-ray diffraction pattern of an acetone adduct of
V-28-3M.
FIG. 2 is a .sup.1 H-NMR spectrum of the acetone adduct of V-28-3M.
FIG. 3 is a UV absorption spectrum of the acetone adduct of V-28-3M.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
The V-28-3M-containing materials used in the present invention include a
solid obtained by drying a reaction solution resulting from
methyl-esterified polyene macrolide V-28-3 produced by Streptomyces arenae
V-28-3 with diazomethane; a precipitate obtained by adding water to said
reaction solution; and a precipitate obtained by the methyl esterification
using a compound other than diazomethane, such as a precipitate obtained
by protecting the amino group of the amino sugar in V-28-3 with a suitable
protecting group and then methyl-esterifying it with, for example, methyl
p-toluenesulfonate, removing the protecting group and adding water to the
reaction solution (PCT/JP 98/02439). Such a precipitate may contain a
large amount of substances derived from the culture or by-products of the
reaction.
In the production of the acetone adduct of V-28-3M, the V-28-3M-containing
material is dissolved in acetone under heating. The dissolution is
conducted at a temperature in the range of 20.degree. C. to the boiling
point of acetone, preferably 50 to 57.degree. C. The concentration is
preferably in the range of 0.1 to 10 g/l, more preferably 2 to 3 g/l. If
necessary, the extraction can be continuously conducted using Soxhlet's
extractor.
The concentration of the obtained solution of V-28-3M in acetone is
adjusted to 2 to 3 g/l by diluting or concentrating the solution under
reduced pressure. The solution is cooled to preferably 10.degree. C. or
below, more preferably 0 to -20.degree. C. The precipitate thus formed has
a high impurity content. The impurities are preferably separated because
they will inhibit the subsequent crystallization of the acetone adduct of
V-28-3M. The separation methods include conventional methods such as
filtration and centrifugation. After the separation of the precipitate
thus formed, the remaining solution is concentrated, if necessary, to
control the concentration of the mother liquor in the range of preferably
1 to 20 g/l, more preferably 2 to 4 g/l. Seed crystals are added to the
liquid and the obtained mixture is cooled to 20.degree. C. or below to
form crystals of the acetone adduct of V-28-3M. The resulting crystals are
filtered or centrifuged, washed with acetone and dried under reduced
pressure to obtain the crystals of the acetone adduct of V-28-3M having an
extremely high purity.
The acetone adduct of V-28-3M thus obtained can be immediately and easily
hydrolyzed by dissolving it in a water containing solvent such as
acetonitrile+water (1:1). The water containing solvent may be any of
water-soluble solvents containing water, such as methanol, ethanol,
isopropyl alcohol and acetonitrile. Water preferably contains 0.01 to 1 M
of a weak acidic salt having a buffering effect such as ammonium acetate,
sodium acetate or sodium phosphate. The acetone adduct of V-28-3M is
dissolved in the water containing solvent to obtain a solution having a
concentration of preferably 0.1 to 10 g/l, more preferably 1 to 2 g/l.
Then the solution is concentrated under reduced pressure to volatilize the
solvent and then V-28-3M is precipitates due to its poor solubility. When
a salt is dissolved in the water containing solvent, the salting-out
effect is also obtained as the solution is concentrated and thus, the
precipitate can be easily obtained. After separating the precipitate
formed during the concentration by filtration, the precipitate is washed
with water or a solvent and then dried under reduced pressure to obtain
pure V-28-3M.
When the purity of V-28-3M-containing material is very low, the solubility
thereof in acetone decreases or the solution remains supersaturated in the
course of the crystallization, which makes the stable crystallization
impossible. Therefore, when the purity of V-28-3M-containing material is
very low, the purity thereof or of the crude precipitate is preferably
increased by the method described below prior to the crystallization.
Namely, the V-28-3M-containing material is dissolved in dimethyl sulfoxide
or dimethylformamide, an organic solvent is added to the obtained solution
to precipitate impurities in the solution and another organic solvent
having a lower polarity is added to the mother liquor to obtain the
intended compound as a precipitate.
Specifically, the V-28-3M-containing material is dissolved in dimethyl
sulfoxide at room temperature to obtain a solution having a concentration
of preferably 1 to 100 g/l, more preferably 10 to 30 g/l. Then a first
organic solvent such as ethanol, isopropyl alcohol, n-butanol, ethyl
acetate or isopropyl acetate is added in an amount of preferably 1 to 20
parts, more preferably 5 to 10 parts, per part of dimethyl sulfoxide to
the solution to precipitate the impurities. After filtration, a second
organic solvent having a lower polarity such as n-hexane, n-heptane or
toluene is added in an amount of preferably 1 to 20 parts, more preferably
2 to 10 parts, per part of dimethyl sulfoxide to the filtrate to
precipitate V-28-3M. The precipitate is collected by filtration, washed
with a small amount of a solvent such as a lower alcohol or an acetic
ester and then dried under reduced pressure. Thus, the impurities having a
hydrophobicity different from that of the intended compound are remarkably
reduced in amount and the precipitate having an improved purity can be
obtained. Dimethyl sulfoxide can be replaced with dimethylformamide.
The following Examples will further illustrate the present invention.
EXAMPLE 1
1-1. Preparation of N-(9-fluorenylmethoxycarbonyl)-V-28-3:
37.38 g (22.92 mmol) of crude V-28-3 was dissolved in 2.4 liters of a
solution of dimethyl sulfoxide/methanol (mixing volume ratio: 9:2).
N-(9-fluorenylmethoxycarbonyloxy)succinimide (38.3 g) was added to the
solution in four portions at room temperature. The reaction solution was
stirred at room temperature for 2 hours and then cooled in an ice bath.
2.4 liters of water was added dropwise to the solution for one hour to
obtain the title compound in the form of a slurry. The title compound was
separated from the slurry, and washed with 300 ml of water and 500 ml of
methanol. The crude precipitate was separated and then dried to obtain
49.28 g (yield: 94%) of crude crystals of the title compound. In the
subsequent methyl esterification reaction, the crude crystal was used
without purification.
1-2. Preparation of N-(9-fluorenylmethoxycarbonyl)-V-28-3M:
13.5 g (9.5 mmol) of crude N-(9-fluorenylmethoxycarbonyl)-V-28-3 prepared
as prepared above was dissolved in 700 ml of a solution of dimethyl
sulfoxide/methanol (mixing volume ratio: 9:2) at room temperature. 7.2 ml
of methyl p-toluenesulfonate and 1.3 g of potassium carbonate were added
to the obtained solution, and the resultant mixture was stirred at
25.degree. C. for 4 hours. Then the reaction solution was cooled to
5.degree. C. and stirred overnight to obtain a reaction solution
containing 11.9 g (yield: 93%) of the title compound. The reaction
solution was subjected to the deprotection reaction in the subsequent step
without isolating the title compound.
1-3. Preparation of V-28-3M:
150 ml of 28% ammonia water (10 v/v% based on the reaction solution) was
added to 1500 ml of mixed solvent of dimethyl sulfoxide/methanol (mixing
ratio: 9/2) containing 25.8 g (19.3 mmol) of
N-(9-fluorenylmethoxycarbonyl)-V-28-3M, prepared as described above, at
25.degree. C., and the obtained mixture was stirred for 2 hours to obtain
the reaction solution containing 17.8 g (yield: 82%) of the title
compound.
1-4. Preparation of acetone adduct of V-28-3M:
Water was added to the reaction solution containing V-28-3M, prepared as
described above, to separate V-28-3M. After separating and drying, 14.8 g
of the obtained crude precipitate containing 59% of V-28-3M was dissolved
in 0.62 liter of dimethyl sulfoxide at room temperature, and then 4.4
liters of ethyl acetate was added dropwise in the resulting solution under
stirring. The precipitate was taken by filtration and 2.6 liters of
n-heptane was added dropwise to the filtrate in the same manner as
described above. The precipitate thus obtained was taken by the
filtration, washed with 0.1 liter of ethyl acetate and dried under reduced
pressure to obtain 8.6 g (recovery rate: 80%) of V-28-3M precipitate
having a purity of 81%.
4.3 g of the precipitate containing 81% of V-28-3M was dissolved in 1.8
liters of acetone at a boiling point (57.degree. C.) in a continuous
extraction tank for 6 hours. The obtained solution was cooled at
-10.degree. C. overnight, and the precipitate thus formed was taken by the
filtration. The mother liquor was concentrated to a volume of 4/5 under
reduced pressure. 30 mg of seed crystals were added to the concentrate.
After cooling at -10.degree. C. overnight, the crystals thus formed were
taken by the filtration, washed with 100 ml of acetone and dried under
reduced pressure to obtain 2.1 g (recovery rate: 60%) of acetone adduct of
V-28-3M having a purity of 98% or above. The crystals thus obtained had
the following physicochemical properties:
1) Appearance: yellow crystals
2) Mass spectrometric analysis: 1167.8 (FABMS [(M+H).sup.+ ])
3) Powder X-ray diffraction: as shown in FIG. 1.
4) .sup.1 H-NMR (determined in DMF-d.sub.7): as shown in FIG. 2
.delta.7.78 ppm (d, J=8.79, aromatic-H), 6.72 (d, J=8.55, aromatic-H),
.delta.3.73 ppm (s, methyl ester), (3.50 (s, acetone adduct part)
8.05 ppm (DMF), 2.94 ppm (DMF), 2.77 ppm (DMF) and 2.15 ppm (acetone) were
solvent signals.
5) UV spectrum (determined in MeOH): as shown in FIG. 3
.lambda..sub.max : 363, 382, 405 nm
From the above-described physicochemical properties, it was confirmed that
the obtained crystals were the acetone adduct wherein the amino sugar
moiety of V-28-3M are condensed with acetone.
EXAMPLE 2
Water was added to the same V-28-3M methyl esterification reaction solution
as that of Example 1 and then the resultant precipitate was separated and
dried to obtain a precipitate containing 49% of V-28-3M. 0.7 g of the
precipitate thus obtained was dissolved in 1.0 liter of acetone at
50.degree. C. for 12 hours. The obtained solution was concentrated to a
volume of 1/12 under reduced pressure and then cooled at -10.degree. C.
overnight. The resultant precipitate was taken by filtration. The mother
liquor was concentrated to a volume of 1/2 under reduced pressure. 2 mg of
seed crystals were added to the concentrate, and the obtained mixture was
cooled at -10.degree. C. overnight. The crystals thus formed were washed
with about 10 ml of acetone and then dried under reduced pressure to
obtain 0.08 g (recovery rate: 24%) of acetone adduct of V-28-3M having a
purity of 98% or higher. In this Example, the purity of the product was
equal to that in Example 1 but the recovery rate was less than that in
Example 1. This fact indicates that the higher the purity of the
V-28-3M-containing material is, the higher the recovery rate is, which is
more useful in the present invention.
EXAMPLE 3
2.1 g of the crystals of the acetone adduct of V-28-3M obtained in Example
1 were dissolved in 1.8 liters of a solution of 50 mM ammonium acetate (pH
5.5)+acetonitrile (1:1) at room temperature, and the obtained solution was
concentrated to a volume of 1/2 under reduced pressure. The precipitates
thus formed were collected by centrifugation, washed with ethanol and
dried under reduced pressure to obtain 1.5 g (yield: 71%) of purified
V-28-3M having a purity of 98% or higher.
The crystal thus obtained had a molecular weight equal to that of V-28-3M
determined by MS spectrum, and the .sup.1 H-NMR pattern of the crystal was
the same as that of V-28-3M.
REFERENTIAL EXAMPLE 1
120 mg of crude precipitates containing 50% of V-28-3M was dissolved in 12
ml of dimethyl sulfoxide at room temperature. 96 ml of isopropyl alcohol
was added dropwise to the obtained solution under stirring. Then, the
precipitates thus formed were taken by filtration, and 108 ml of n-hexane
was added dropwise to the filtrate in the same manner as that described
above. The precipitates thus formed were taken by filtration, washed with
10 ml of n-hexane and dried under reduced pressure to obtain 41 mg of
precipitates of V-28-3M having a purity of 80%.
REFERENTIAL EXAMPLE 2
350 mg of crude precipitates containing 36% of V-28-3M was dissolved in 6
ml of dimethylformamide at room temperature. 12 ml of ethyl acetate was
added dropwise to the obtained solution under stirring. Then, the
precipitates thus formed were taken by filtration, and 18 ml of n-heptane
was added dropwise to the filtrate in the same manner as that described
above. The precipitates thus formed were taken by filtration, washed with
5 ml of ethyl acetate and dried under reduced pressure to obtain 118 mg of
precipitates of V-28-3M having a purity of 80%.
COMPARATIVE EXAMPLE 1
The HPLC purification was conventionally conducted as follows:
Water was added to a reaction solution obtained by the methyl
esterification reaction of V-28-3B as described in JP-Kokai 5-59084. After
the separation, the precipitate was dried to obtain 2.5 g of the
precipitate containing 50% of V-28-3M, which was then dissolved in 42 ml
of dimethyl sulfoxide. 83 ml of a mixture of 50 mM aqueous solution of
acetic acid+acetonitrile (1:1) was added to the obtained solution. Then
125 ml of a mixture of 50 mM ammonium acetate (pH 5.7)+acetonitrile
(52:48) was added to the obtained mixture and the loading solution was
prepared by filtrating out the solid material. 250 ml of the loading
solution was passed through a preparative, reversed phase HPLC column YMC
ODS R-3101-20, R-3105-50 (100.phi.x(100+500)) at a rate of 30 ml/min, and
eluted using 10 liters of a mixture of 50 mM ammonium acetate (pH
5.7)+acetonitrile (52:48) at a rate of 300 ml/min. About 500 ml of V-28-3M
fraction taken according to 280 nm UV absorption pattern was concentrated
to a volume of about 1/2 under reduced pressure, and then left to stand at
4.degree. C. overnight. Precipitates thus formed were collected by
centrifugation, washed with water twice and then freeze-dried. 0.6 g
(recovery rate: 48%) of V-28-3M having a purity of 86% was obtained.
In this method, the required amount of the solvent is more than 10 times as
large as that required for processing the same amount of the starting
material according to the acetone adduct crystallization method of Example
1. The productivity in this method was thus extremely low. Further
enlargement of the column is required to increase the amount of the
preparation, because it is not expected that the loading amount can be
further increased for the column used in this method. However, the scale
up of the column is technically difficult in general. In addition, the
resin packed in the column is very easily degradated when this starting
material is used. Since the filling material itself is expensive, the
frequent exchange thereof and the large amount of solvent used cause an
increase in the cost.
For the reasons described above, the purification with the column has
problems in the productivity and the cost.
Thus, the crystallization rate of V-28-3M can be remarkably improved by
forming the acetone adduct thereof according to the present invention, and
the high purification can be attained without employing the reversed phase
HPLC or counter current distribution method wherein a large amount of the
solvent is necessitated. Thus, the present invention provides the method
of producing V-28-3M using the crystallization process wherein
purification can be conducted with relatively smaller amount of the
solvent than that of HPLC and the scale up is easier than the column
chromatography process, as well as the method of purifying V-28-3M
suitable for industrial application wherein a conventional apparatus can
be used, which is advantageous in the cost.
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